Traveling or living in certain conditions or areas
Poverty and substance abuse
http://www.mayoclinic.org/diseases-conditions/tuberculosis/basics/risk-factors/con-20021761 *Disease Information: Mycobacterium tuberculosis is an organism that causes the well-known disease, tuberculosis (TB). Specifically, M. tuberculosis is pathogenic towards humans and usually attacks the lungs, however can infect any part of the body. It is an airborne pathogen that can spread from one person to another. TB is one of the leading causes of death due to an infectious disease. M. tuberculosis is able to avoid intercellular killing and enter macrophages, thus a reason for its prevalence and severity. There are two forms of TB: latent TB infection and TB disease, where the M. bacterium is inactive in the body and does not cause harm or the bacteria is active and the immune system cannot fight it off, respectively. The target focused on, serine threonine kinase protein B is essential for cell division and cell shape since it is a transmembrane protein. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC96941/ http://www.cdc.gov/tb/topic/basics/default.htm
Query 9 DRYELGEILGFGGMSEVHLARDLRLHRDVAVKVLRADLARDPSFYLRFRREAQNAAALNH 68 D Y + ++G G L + ++ A+K +R L + S R+EA A + H Sbjct 2 DDYMVLRMIGEGSFGRALLVQHESSNQMFAMKEIR--LPKSFSNTQNSRKEAVLLAKMKH 59
Query 69 PAIVAVYDTGEAETPAGPLPYIVMEYVDGVTL-RDIVHTEGPMTPKRAI-EVIADACQAL 126 P IVA ++ EAE G L YIVMEY DG L + I +G + P+ I C + Sbjct 60 PNIVAFKESFEAE---GHL-YIVMEYCDGGDLMQKIKQQKGKLFPEDMILNWFTQMCLGV 115
Query 127 NFSHQNGIIHRDVKPANIMISATNAVKVMDFGIARAIADSGNSVTQTAAVIGTAQYLSPE 186 N H+ ++HRD+K NI ++ VK+ DFG AR ++ N + +GT Y+ PE Sbjct 116 NHIHKKRVLHRDIKSKNIFLTQNGKVKLGDFGSARLLS---NPMAFACTYVGTPYYVPPE 172
Query 187 QARGDSVDARSDVYSLGCVLYEVLTGEPPFTGDSPVSVAYQHVREDPIPPSARHEGLSAD 246 + +SD++SLGC+LYE+ T + PF +S ++ + V + I P H S + Sbjct 173 IWENLPYNNKSDIWSLGCILYELCTLKHPFQANSWKNLILK-VCQGCISPLPSH--YSYE 229
Information found for another bacteria (Corynebacterium glutamicum): Assays carried out with a buffer of Tris-HCl, diothreitol, MgCl2 and EDTA)
In Vitro Kinase Assays—In vitro phosphorylation of each recombinant kinase (0.5 μg) was carried out for 30 min at 37 °C in a reaction mixture (20 μl) containing buffer P (25 mM Tris-HCl, pH 7.0, 1 mM dithiothreitol, 5 mM MgCl2, 1 mM EDTA) with 200 μCi/ml [γ-33P]ATP corresponding to 65 nM (3000 Ci/mmol). After incubation, the reaction was stopped by adding sample buffer and heating the mixture at 100 °C for 5 min.
Competent cells of E. coliBL21(DE3) were prepared according to the CaCl2 method (30) and were transformed by the heat shock method for 2 min at 42°C with 100 ng of pYA102. The transformed E. coli cells were then plated onto LB agar supplemented with ampicillin (100 μg/ml). Single colonies were inoculated into 5 ml of LB broth also containing ampicillin (100 μg/ml). After overnight incubation at 37°C with shaking, the individual cultures were diluted 1:100 in the same medium and incubation was continued at 37°C with shaking. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM when the optical density at 600 nm reached 0.6. Cultures were centrifuged at 5,000 × g for 15 min at room temperature, and pellets were lysed in B-Per (Pierce) bacterial protein extraction reagent. Proteins were separated by centrifugation (15,000 × g, 4°C, 15 min) into soluble and insoluble fractions. PknB inclusion bodies contained in the insoluble fractions were purified from E. colimembrane proteins by washing in a solution of 10% B-Per reagent and centrifugation (45,000 × g, 90 min, 4°C). PknB was separated by sodium dodecyl sulfate–7.5% polyacrylamide electrophoresis (SDS-PAGE) and stained with Coomassie blue or transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The N-terminal amino acid sequence was verified after electrophoresis of samples in SDS-PAGE gels and electroblotting onto PVDF membranes. Edman degradation was performed, and the sequence of the first 10 amino acids from the NH2 terminus was determined at the University of British Columbia Protein Sequencing Laboratory. In order to obtain soluble protein, PknB inclusion bodies were resuspended in 1× phosphate-buffered saline (PBS) (pH 7.4) and slowly added drop-wise to a solution of 16 M urea and 2 M dithiothreitol (DTT) to make a final concentration of 8 M urea and 1 M DTT. Soluble PknB was then dialyzed via a Spectra/Por 8000 cellulose membrane (VWR Scientific) against 200 volumes of 1× Tris-buffered saline (pH 7.4) at 4°C for 16 to 24 h. The sample was then centrifuged for 15 min at 4°C and 15,000 × g (Baxter), and approximately 20 mg of protein was loaded onto a 50-ml Macro-Prep SE agarose size-exclusion column (Bio-Rad), which was used as a desalting column. Proteins were eluted over time at 4°C with a size-exclusion buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM DTT, and 0.01 mM EDTA (18). The purity of PknB was tested by subjecting samples to SDS-PAGE followed by Coomassie blue staining. SDS-PAGE gels were prepared by the method of Laemmli (17). The gels were stained with Coomassie blue R-250 or silver stain. Protein concentrations were determined by the Bradford protein assay reagent
Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence: MTTPSHLSDRYELGEILGFGGMSEVHLARDLRLHRDVAVKVLRADLARDPSFYLRFRREA QNAAALNHPAIVAVYDTGEAETPAGPLPYIVMEYVDGVTLRDIVHTEGPMTPKRAIEVIA DACQALNFSHQNGIIHRDVKPANIMISATNAVKVMDFGIARAIADSGNSVTQTAAVIGTA QYLSPEQARGDSVDARSDVYSLGCVLYEVLTGEPPFTGDSPVSVAYQHVREDPIPPSARH EGLSADLDAVVLKALAKNPENRYQTAAEMRADLVRVHNGEPPEAPKVLTDAERTSLLSSA AGNLSGPRTDPLPRQDLDDTDRDRSIGSVGRWVAVVAVLAVLTVVVTIAINTFGGITRDV QVPDVRGQSSADAIATLQNRGFKIRTLQKPDSTIPPDHVIGTDPAANTSVSAGDEITVNV STGPEQREIPDVSTLTYAEAVKKLTAAGFGRFKQANSPSTPELVGKVIGTNPPANQTSAI TNVVIIIVGSGPATKDIPDVAGQTVDVAQKNLNVYGFTKFSQASVDSPRPAGEVTGTNPP AGTTVPVDSVIELQVSKGNQFVMPDLSGMFWVDAEPRLRALGWTGMLDKGADVDAGGSQH NRVVYQNPPAGTGVNRDGIITLRFGQ *Length of your protein in Amino Acids: 626 amino acids Molecular Weight of your protein in kiloDaltons: 66.51 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 11,700 M−1 cm−1 http://jb.asm.org/content/191/13/4056.full TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized): Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to. Primer design results for 'tail' primers (this is just 2 sequences):
*NCBI Gene #: 887072
http://www.ncbi.nlm.nih.gov/gene/887072
*Protein ID: 5.08
*Organism: Mycobacterium tuberculosis
Etiologic Risk Group:
- Weakened immune system
- Traveling or living in certain conditions or areas
- Poverty and substance abuse
http://www.mayoclinic.org/diseases-conditions/tuberculosis/basics/risk-factors/con-20021761*Disease Information:
Mycobacterium tuberculosis is an organism that causes the well-known disease, tuberculosis (TB). Specifically, M. tuberculosis is pathogenic towards humans and usually attacks the lungs, however can infect any part of the body. It is an airborne pathogen that can spread from one person to another. TB is one of the leading causes of death due to an infectious disease.
M. tuberculosis is able to avoid intercellular killing and enter macrophages, thus a reason for its prevalence and severity. There are two forms of TB: latent TB infection and TB disease, where the M. bacterium is inactive in the body and does not cause harm or the bacteria is active and the immune system cannot fight it off, respectively. The target focused on, serine threonine kinase protein B is essential for cell division and cell shape since it is a transmembrane protein.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC96941/
http://www.cdc.gov/tb/topic/basics/default.htm
Link to TDR Targets page (if present):
http://www.tdrtargets.org/targets/view?gene_id=6795
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
https://www.patricbrc.org/portal/portal/patric/Feature?cType=feature&cId=PATRIC.1354165.3.JLAV01000005.CDS.72140.74056.rev
Essentiality of this protein: Essential for cell survival because it plays a critical role in regulating cell division and cell wall synthesis. Essentiality was tested in experiments to determine the purpose of serine/threonine PKNB in M. tuberculosis cells.
http://www.jbc.org/content/289/20/13858.long
Multimer biological unit.
Complex protein: not complex
Similarity to human:
D Y + ++G G L + ++ A+K +R L + S R+EA A + H
Sbjct 2 DDYMVLRMIGEGSFGRALLVQHESSNQMFAMKEIR--LPKSFSNTQNSRKEAVLLAKMKH 59
Query 69 PAIVAVYDTGEAETPAGPLPYIVMEYVDGVTL-RDIVHTEGPMTPKRAI-EVIADACQAL 126
P IVA ++ EAE G L YIVMEY DG L + I +G + P+ I C +
Sbjct 60 PNIVAFKESFEAE---GHL-YIVMEYCDGGDLMQKIKQQKGKLFPEDMILNWFTQMCLGV 115
Query 127 NFSHQNGIIHRDVKPANIMISATNAVKVMDFGIARAIADSGNSVTQTAAVIGTAQYLSPE 186
N H+ ++HRD+K NI ++ VK+ DFG AR ++ N + +GT Y+ PE
Sbjct 116 NHIHKKRVLHRDIKSKNIFLTQNGKVKLGDFGSARLLS---NPMAFACTYVGTPYYVPPE 172
Query 187 QARGDSVDARSDVYSLGCVLYEVLTGEPPFTGDSPVSVAYQHVREDPIPPSARHEGLSAD 246
+ +SD++SLGC+LYE+ T + PF +S ++ + V + I P H S +
Sbjct 173 IWENLPYNNKSDIWSLGCILYELCTLKHPFQANSWKNLILK-VCQGCISPLPSH--YSYE 229
Query 247 LDAVVLKALAKNPENR 262
L +V + +NP +R
Sbjct 230 LQFLVKQMFKRNPSHR 245
http://blast.ncbi.nlm.nih.gov/Blast.cgi
Druggable Target: 0.7
*EC#: 2.7.11.1
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/enzyme.php?ecno=2.7.11.1
Enzyme Assay information: In Vitro Kinase Assays
Information found for another bacteria (Corynebacterium glutamicum): Assays carried out with a buffer of Tris-HCl, diothreitol, MgCl2 and EDTA)
In Vitro Kinase Assays—In vitro phosphorylation of each recombinant kinase (0.5 μg) was carried out for 30 min at 37 °C in a reaction mixture (20 μl) containing buffer P (25 mM Tris-HCl, pH 7.0, 1 mM dithiothreitol, 5 mM MgCl2, 1 mM EDTA) with 200 μCi/ml [γ-33P]ATP corresponding to 65 nM (3000 Ci/mmol). After incubation, the reaction was stopped by adding sample buffer and heating the mixture at 100 °C for 5 min.
http://www.jbc.org/content/283/26/18099.long
http://www.ncbi.nlm.nih.gov/pubmed/18442973
--- List cost and quantity of substrate reagents, supplier, and catalog #
http://www.perkinelmer.com/resources/technicalresources/applicationsupportknowledgebase/radiometric/invitro_kinase.xhtml#Radioactiveinvitrokinaseassays-Productsandcatalognumbers
Structure:
-- PDB # 1MRU
Current Inhibitors: ATP kinase inhibitorshttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158675/
Expression Information: Expressed in bacteria cells, such as Corynebacterium glutamicum.
Purification Method:
Method used in an experiment by Yosseff Av Gay:
Competent cells of E. coliBL21(DE3) were prepared according to the CaCl2 method (30) and were transformed by the heat shock method for 2 min at 42°C with 100 ng of pYA102. The transformed E. coli cells were then plated onto LB agar supplemented with ampicillin (100 μg/ml). Single colonies were inoculated into 5 ml of LB broth also containing ampicillin (100 μg/ml). After overnight incubation at 37°C with shaking, the individual cultures were diluted 1:100 in the same medium and incubation was continued at 37°C with shaking. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM when the optical density at 600 nm reached 0.6. Cultures were centrifuged at 5,000 × g for 15 min at room temperature, and pellets were lysed in B-Per (Pierce) bacterial protein extraction reagent. Proteins were separated by centrifugation (15,000 × g, 4°C, 15 min) into soluble and insoluble fractions. PknB inclusion bodies contained in the insoluble fractions were purified from E. colimembrane proteins by washing in a solution of 10% B-Per reagent and centrifugation (45,000 × g, 90 min, 4°C). PknB was separated by sodium dodecyl sulfate–7.5% polyacrylamide electrophoresis (SDS-PAGE) and stained with Coomassie blue or transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The N-terminal amino acid sequence was verified after electrophoresis of samples in SDS-PAGE gels and electroblotting onto PVDF membranes. Edman degradation was performed, and the sequence of the first 10 amino acids from the NH2 terminus was determined at the University of British Columbia Protein Sequencing Laboratory. In order to obtain soluble protein, PknB inclusion bodies were resuspended in 1× phosphate-buffered saline (PBS) (pH 7.4) and slowly added drop-wise to a solution of 16 M urea and 2 M dithiothreitol (DTT) to make a final concentration of 8 M urea and 1 M DTT. Soluble PknB was then dialyzed via a Spectra/Por 8000 cellulose membrane (VWR Scientific) against 200 volumes of 1× Tris-buffered saline (pH 7.4) at 4°C for 16 to 24 h. The sample was then centrifuged for 15 min at 4°C and 15,000 × g (Baxter), and approximately 20 mg of protein was loaded onto a 50-ml Macro-Prep SE agarose size-exclusion column (Bio-Rad), which was used as a desalting column. Proteins were eluted over time at 4°C with a size-exclusion buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM DTT, and 0.01 mM EDTA (18). The purity of PknB was tested by subjecting samples to SDS-PAGE followed by Coomassie blue staining. SDS-PAGE gels were prepared by the method of Laemmli (17). The gels were stained with Coomassie blue R-250 or silver stain. Protein concentrations were determined by the Bradford protein assay reagent
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC96941/
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence:
MTTPSHLSDRYELGEILGFGGMSEVHLARDLRLHRDVAVKVLRADLARDPSFYLRFRREA
QNAAALNHPAIVAVYDTGEAETPAGPLPYIVMEYVDGVTLRDIVHTEGPMTPKRAIEVIA
DACQALNFSHQNGIIHRDVKPANIMISATNAVKVMDFGIARAIADSGNSVTQTAAVIGTA
QYLSPEQARGDSVDARSDVYSLGCVLYEVLTGEPPFTGDSPVSVAYQHVREDPIPPSARH
EGLSADLDAVVLKALAKNPENRYQTAAEMRADLVRVHNGEPPEAPKVLTDAERTSLLSSA
AGNLSGPRTDPLPRQDLDDTDRDRSIGSVGRWVAVVAVLAVLTVVVTIAINTFGGITRDV
QVPDVRGQSSADAIATLQNRGFKIRTLQKPDSTIPPDHVIGTDPAANTSVSAGDEITVNV
STGPEQREIPDVSTLTYAEAVKKLTAAGFGRFKQANSPSTPELVGKVIGTNPPANQTSAI
TNVVIIIVGSGPATKDIPDVAGQTVDVAQKNLNVYGFTKFSQASVDSPRPAGEVTGTNPP
AGTTVPVDSVIELQVSKGNQFVMPDLSGMFWVDAEPRLRALGWTGMLDKGADVDAGGSQH
NRVVYQNPPAGTGVNRDGIITLRFGQ
*Length of your protein in Amino Acids: 626 amino acids
Molecular Weight of your protein in kiloDaltons: 66.51 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
11,700 M−1 cm−1
http://jb.asm.org/content/191/13/4056.full
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
*CDS Gene Sequence:
atgaccaccccttcccacctgtccgaccgctacgaacttggcgaaatccttggatttggg
ggcatgtccgaggtccacctggcccgcgacctccggttgcaccgcgacgttgcggtcaag
gtgctgcgcgctgatctagcccgcgatcccagtttttaccttcgcttccggcgtgaggcg
caaaacgccgcggcattgaaccaccctgcaatcgtcgcggtctacgacaccggtgaagcc
gaaacgcccgccgggccattgccctacatcgtcatggaatacgtcgacggcgttaccctg
cgcgacattgtccacaccgaagggccgatgacgcccaaacgcgccatcgaggtcatcgcc
gacgcctgccaagcgctgaacttcagtcatcagaacggaatcatccaccgtgacgtcaag
ccggcgaacatcatgatcagcgcgaccaatgcagtaaaggtgatggatttcggcatcgcc
cgcgccattgccgacagcggcaacagcgtgacccagaccgcagcagtgatcggcacggcg
cagtacctgtcacccgaacaggcccggggtgattccgtcgacgcccgatccgatgtctat
tccttgggctgtgttctttatgaagtcctcaccggggagccacctttcaccggcgactca
cccgtctcggttgcctaccaacatgtgcgcgaagacccgatcccaccttcggcgcggcac
gaaggcctctccgccgacctggacgccgtcgttctcaaggcgctggccaaaaatccggaa
aaccgctatcagacagcggcggagatgcgcgccgacctggtccgcgtgcacaacggtgag
ccgcccgaggcgcccaaagtgctcaccgatgccgagcggacctcgctgctgtcgtctgcg
gccggcaaccttagcggtccgcgcaccgatccgctaccacgccaggacttagacgacacc
gaccgtgaccgcagcatcggttcggtgggccgttgggttgcggtggtcgccgtgctcgct
gtgctgaccgtcgtggtaaccatcgccatcaacacgttcggcggcatcacccgcgacgtt
caagttcccgacgttcggggtcaatcctccgccgacgccatcgccacactgcaaaaccgg
ggcttcaaaatccgcaccttgcagaagccggactcgacaatcccaccggaccacgttatc
ggcaccgacccggccgccaacacgtcggtgagtgcaggcgacgagatcacagtcaacgtg
tccaccggacccgagcaacgcgaaatacccgacgtctccacgctgacatacgccgaagcg
gtcaagaaactgactgccgccggattcggccgcttcaagcaagcgaattcgccgtccacc
ccggaactggtgggcaaggtcatcgggaccaacccgccagccaaccagacgtcggccatc
accaatgtggtcatcatcatcgttggctctggtccggcgaccaaagacattcccgatgtc
gcgggccagaccgtcgacgtggcgcagaagaacctcaacgtctacggcttcaccaaattc
agtcaggcctcggtggacagcccccgtcccgccggcgaggtgaccggcaccaatccaccc
gcaggcaccacagttccggtcgattcagtcatcgaactacaggtgtccaagggcaaccaa
ttcgtcatgcccgacctatccggcatgttctgggtcgacgccgaaccacgattgcgcgcg
ctgggctggaccgggatgctcgacaaaggggccgacgtcgacgccggtggctcccaacac
aaccgggtcgtctatcaaaacccgccggcggggaccggcgtcaaccgggacggcatcatc
acgctgaggttcggccagtag
*GC% Content for gene: 63.3%
http://www.genomicsplace.com/cgi-bin/gc_calculator.pl
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):